A nucleic acid detection method and kit for distinguishing pathogen DNA / RNA

Abstract

The application discloses a nucleic acid detection method and kit for distinguishing pathogen DNA / RNA, and a section of artificial sequence is introduced at the 5' end of a primer to improve the specificity of the primer, the artificial sequence is not complementary to a template, and the artificial sequence is complementary to the 3' end sequence of the primer to form a stem loop structure. When the template is not completely complementary to the 3' end of the primer, the primer exists in the form of the stem loop structure, and when the template is completely complementary to the 3' end of the primer, the stem loop structure is unbound to perform reverse transcription. When RNA is amplified, the synthesized cDNA is provided with the artificial sequence, the primer is completely complementary to the cDNA during amplification, 7-11 bases at the 3' end of the primer are complementary to the template, and deoxyuracil is used to replace deoxythymine base at the 3' end of the primer. According to the application, the Tm value of the primer binding is relatively low when DNA is amplified under a certain annealing temperature, so that the DNA cannot be amplified, and when RNA is amplified, the 5' end of the reverse transcription primer is provided with the artificial sequence, so that the synthesized cDNA is provided with the artificial sequence, the primer can be completely complementary to the cDNA during amplification, and the RNA can be accurately detected.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a nucleic acid detection method and kit for distinguishing pathogen DNA / RNA. Background Technology

[0002] Pathogens are infectious microorganisms or biological vectors that can cause disease after invading a host. These include bacteria, fungi, viruses, and viroids. Pathogens can be carried in food, water, and soil. Prolonged exposure to pathogens or ingestion of food or water contaminated with pathogens can lead to illness. Pathogen infections can cause various diseases affecting the respiratory, digestive, and reproductive tracts. Timely detection of pathogens is crucial for disease treatment.

[0003] Currently, most pathogen diagnostic methods include culture, immunological, and molecular biological methods. Culture is the routine laboratory diagnostic method for pathogens. Traditional culture methods have high specificity and sensitivity, but clinical testing is time-consuming (at least 24-48 hours), cumbersome, and the positive rate is affected by various factors, making it unsuitable for large-scale testing. Serological methods, due to the window period, have a diagnostic lag and cannot accurately reflect the current infection status. Although they have high specificity, their sensitivity is insufficient, resulting in a low detection rate. Molecular diagnostic methods include gene sequencing and nucleic acid detection. Gene sequencing is expensive, complex, and time-consuming, making it unsuitable for clinical testing. Pathogen nucleic acid fluorescence PCR detection has advantages such as speed, accuracy, and high sensitivity. However, because DNA structure is stable, it can remain positive for a long time after bacterial death, making it impossible to determine the specific stage of pathogen infection, leading to overtreatment. RNA has a short half-life and degrades rapidly after bacterial death. Detecting pathogen RNA alone can differentiate bacterial activity. Furthermore, because multiple copies of RNA exist in the cells of non-viral pathogenic microorganisms, it offers higher sensitivity and accuracy compared to DNA-targeting techniques such as PCR. RNA not only possesses the advantages of rapid and highly sensitive nucleic acid detection but can also filter out dead bacteria, providing more precise detection and making it suitable for clinical screening and diagnosis.

[0004] For RNA detection technology, one of the main challenges is how to eliminate DNA interference in the sample, maintain RNA stability, and ensure sensitivity and accuracy.

[0005] RT-qPCR is a mature molecular detection technology that was applied to clinical testing relatively early and is widely used in clinical practice. However, it has certain limitations in the detection of some RNAs. The main reasons are: ① DNA digestion inevitably leads to RNA loss, which reduces the sensitivity of the detection; ② When the elimination is incomplete, the residual DNA and RNA are amplified together, affecting the accuracy of quantification.

[0006] Currently, the most commonly used technique for RNA detection abroad is transcription-mediated amplification (TMA), introduced by Gen-Probe in 1995. This technique uses MMLV reverse transcriptase and T7 RNA polymerase to amplify RNA under isothermal conditions. The amplified RNA product is then qualitatively detected using a matching hybridization protection assay. While this technique can detect RNA and has high sensitivity, the endpoint TMA method is prone to contamination by amplified RNA, often resulting in false positives and significantly limiting its application in clinical testing.

[0007] Shanghai Rendu's Simultaneous Amplification and Testing (SAT) technology uses a similar amplification principle to TMA, but SAT introduces molecular beacons during the detection process, eliminating the need for post-amplification cap opening for endpoint testing. This reduces the possibility of contamination to some extent. However, this technology is unstable and susceptible to amplification failure due to various factors. Furthermore, it requires adding T7 RNA polymerase after reverse transcription, making the process somewhat cumbersome. Due to limitations in the number of fluorescence channels, no multiplex detection products are currently available on the market.

[0008] Shanghai Kehua Bioengineering Co., Ltd. (Application Publication No. CN 101948908 A) proposed a patent that modifies primers based on RT-PCR by introducing an auxiliary oligonucleotide sequence. This sequence is complementary to RNA at its 3' end and contains an artificial sequence at its 5' end. During reverse transcription of the RNA sample, the artificial sequence from the primers is introduced into the cDNA. During amplification, the artificial sequence is completely complementary to the cDNA, allowing for normal amplification. However, this method has certain limitations, requiring the auxiliary oligonucleotide sequence to be reverse transcribed first, followed by amplification using the artificial sequence, making the process relatively cumbersome.

[0009] Therefore, there is an urgent need in this field for an accurate, sensitive, and convenient method for detecting pathogen nucleic acids, which can specifically detect RNA while maintaining high sensitivity. To address this need, this invention establishes a PCR method and kit for specific RNA detection through unique primer design. Summary of the Invention

[0010] To overcome the problems of poor sensitivity and inability to distinguish between dead and live bacteria in existing DNA amplification techniques, and low efficiency, susceptibility to contamination, poor stability, cumbersome operation, and high cost of RNA amplification technology, this invention establishes a nucleic acid detection method and kit for distinguishing pathogen DNA / RNA based on good stability and high amplification efficiency.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] This invention first provides a nucleic acid detection method for distinguishing pathogen DNA / RNA. The nucleic acid detection method includes: introducing an artificial sequence at the 5' end of a primer to improve primer specificity; the artificial sequence is not complementary to the template, but is complementary to the 3' end sequence of the primer to form a stem-loop structure; when the template and the 3' end of the primer are not completely complementary, the primer exists in a stem-loop structure; when the template and the 3' end of the primer are completely complementary, the stem-loop structure unwinds and reverse transcription is performed; during RNA amplification, the synthesized cDNA carries the artificial sequence, and during amplification, the primer and cDNA are completely complementary.

[0013] As a preferred embodiment of the present invention, the 3' end of the primer has 7-11 bases that are complementary to the template.

[0014] As a preferred embodiment of the present invention, the 3' end of the primer uses deoxyuracil instead of deoxythymidine bases.

[0015] In this invention, primers are modified based on the relatively mature RT-qPCR technology to achieve specific RNA detection without a DNA digestion step. Inspired by the principle that different primer binding Tm values ​​at a given annealing temperature result in different amplification efficiencies, this invention has found through experimentation that when the 3' end of the primer has only 7-11 complementary bases to the template, DNA amplification is virtually nonexistent at annealing temperatures of 58-64℃. Experiments have shown that the fewer the number of bound bases, the lower the DNA amplification efficiency. While only 7-11 complementary bases at the 3' end have little impact on transcription, a slight decrease in reverse transcription efficiency occurs with further reductions in the number of bound bases. Research has shown that deoxyuridine can be inserted into oligonucleotides to increase the melting point temperature of the double strand, thereby increasing double-strand stability and reducing the impact of a low base count on transcription efficiency. Therefore, to prevent DNA amplification without affecting reverse transcription efficiency, the inventors used deoxyuridine instead of deoxythymine, replacing deoxythymine with primers having only 7-11 complementary bases at the 3' end, thus mitigating the impact of a low base count on transcription efficiency.

[0016] Simultaneously, an artificial sequence is introduced at the 5' end. To minimize DNA amplification efficiency and improve primer specificity, this artificial sequence must have the following characteristics:

[0017] 1) This sequence segment is not complementary to the template;

[0018] 2) This artificial sequence can be complementary to the 3' end sequence of the primer to form a simple stem-loop structure. When the template and the 3' end of the primer are not completely complementary, the primer exists as a stem-loop structure. When the template and the 3' end of the primer are completely complementary, the stem-loop structure unwinds and reverse transcription is performed.

[0019] At this point, under a certain annealing temperature, DNA cannot be amplified because the Tm value of primer binding is low. However, when amplifying RNA, the artificial sequence at the 5' end of the reverse transcription primer causes the synthesized cDNA to carry this artificial sequence. During amplification, the primer can be completely complementary to the cDNA, allowing for accurate detection of RNA.

[0020] As a preferred embodiment of the present invention, the pathogen is a Gram-positive pathogen, a Gram-negative pathogen, or a fungal pathogen.

[0021] In a preferred embodiment of the present invention, when the pathogen is a Gram-positive pathogen, the sequence of the downstream primer is shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, the sequence of the upstream primer is shown in SEQ ID NO: 2, and the sequence of the probe is shown in SEQ ID NO: 7.

[0022] In a preferred embodiment of the present invention, when the pathogen is a Gram-negative pathogen, the sequence of the downstream primer is shown in SEQ ID NO: 10, the sequence of the upstream primer is shown in SEQ ID NO: 9, and the sequence of the probe is shown in SEQ ID NO: 11.

[0023] In a preferred embodiment of the present invention, when the pathogen is a fungal pathogen, the sequence of the downstream primer is shown in SEQ ID NO: 13, the sequence of the upstream primer is shown in SEQ ID NO: 14, and the sequence of the probe is shown in SEQ ID NO: 15.

[0024] The present invention also provides a nucleic acid detection kit that includes the above-mentioned primers to distinguish between pathogen DNA / RNA.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1) This invention introduces an artificial sequence at the 5' end of the primer in order to minimize DNA amplification efficiency and improve primer specificity.

[0027] 2) This invention uses deoxyuracil instead of deoxythymine, which has fewer bases than the template complementary bases at the 3' end of the primer (only 7-11 bases), to reduce transcription efficiency.

[0028] 3) Under a certain annealing temperature, the present invention prevents DNA from being amplified due to the low Tm value of primer binding. However, when amplifying RNA, the artificial sequence at the 5' end of the reverse transcription primer causes the synthesized cDNA to carry the artificial sequence. During amplification, the primer can be completely complementary to the cDNA, and RNA can be accurately detected. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the principle of the present invention.

[0030] Figure 2 This describes the amplification of different samples using the modified primers in the RT-qPCR reaction system.

[0031] Figure 3 Example 1 is in 10 4 Amplification curves at copies / ml GBS.

[0032] Figure 4 Example 1 is in 10 3 Amplification curves at copies / ml GBS.

[0033] Figure 5 Example 2 is in 10 3 Amplification curves at copies / ml NG.

[0034] Figure 6 Example 2 is in 10 2 Amplification curves at copies / ml NG.

[0035] Figure 7 Example 3 is in 10 4 Amplification curves of Candida albicans copies / ml.

[0036] Figure 8 Example 3 is in 10 3 Amplification curves of Candida albicans copies / ml. Detailed Implementation

[0037] To facilitate understanding of the technical means, creative features, objectives, and effects of this invention, the invention is further described below with reference to specific embodiments. However, the following embodiments are merely preferred embodiments of this invention and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the scope of protection of this invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0038] See Figure 1 and Figure 2 This invention provides a nucleic acid detection method for distinguishing pathogen DNA / RNA. The nucleic acid detection method includes: introducing an artificial sequence at the 5' end of a primer to improve primer specificity; the artificial sequence is not complementary to the template, but is complementary to the 3' end sequence of the primer to form a stem-loop structure; when the template and the 3' end of the primer are not completely complementary, the primer exists in a stem-loop structure; when the template and the 3' end of the primer are completely complementary, the stem-loop structure unwinds and reverse transcription is performed; during RNA amplification, the synthesized cDNA carries the artificial sequence, and during amplification, the primer and cDNA are completely complementary.

[0039] The 3' end of the primer has 7-11 bases that are complementary to the template.

[0040] The 3' end of the primer uses deoxyuracil instead of deoxythymidine.

[0041] Inspired by the principle that different primer binding Tm values ​​at a given annealing temperature result in different amplification efficiencies, this invention, through practical experience, found that when the primer's 3' end is only 7-11 complementary bases to the template, DNA amplification is essentially nonexistent at annealing temperatures of 58-64℃. Experiments showed that the fewer the number of bound bases, the lower the DNA amplification efficiency. While only 7-11 complementary bases at the 3' end have little impact on transcription, a slight decrease in reverse transcription efficiency occurs with further reductions in the number of bound bases. Research has shown that deoxyuridine can be inserted into oligonucleotides to increase the melting point of the double strand, thereby increasing its stability and reducing the impact of a low base count on transcription efficiency. Therefore, to prevent DNA amplification without affecting reverse transcription efficiency, the inventors used deoxyuridine instead of deoxythymine, replacing deoxythymine with primers that only have 7-11 complementary bases at the 3' end to the template, thus mitigating the impact of a low base count on transcription efficiency.

[0042] Simultaneously, an artificial sequence is introduced at the 5' end. To minimize DNA amplification efficiency and improve primer specificity, this artificial sequence must have the following characteristics:

[0043] 1) This sequence segment is not complementary to the template;

[0044] 2) This artificial sequence can be complementary to the 3' end sequence of the primer to form a simple stem-loop structure. When the template and the 3' end of the primer are not completely complementary, the primer exists as a stem-loop structure. When the template and the 3' end of the primer are completely complementary, the stem-loop structure unwinds and reverse transcription is performed.

[0045] At this point, under a certain annealing temperature, DNA cannot be amplified because the Tm value of primer binding is low. However, when amplifying RNA, the artificial sequence at the 5' end of the reverse transcription primer causes the synthesized cDNA to carry this artificial sequence. During amplification, the primer can be completely complementary to the cDNA, allowing for accurate detection of RNA.

[0046] Example 1

[0047] RNA detection of Gram-positive pathogens

[0048] This example uses Group B Streptococcus RNA detection:

[0049] Group B Streptococcus (GBS), also known as agalactococcus, is a facultative anaerobic Gram-positive coccus. Based on differences in the bacterial capsular polysaccharide, GBS can be classified into Ia, Ib, Ic, II, III, IV, V, VI, VII, VIII, and IX. GBS can colonize the digestive and reproductive tracts intermittently, transiently, or persistently. GBS colonization in pregnant women refers to a positive GBS culture from vaginal, rectal, or perianal samples taken during pregnancy. GBS is an opportunistic pathogen; under certain conditions, it can transform from a colonizing state to a pathogenic one, leading to invasive GBS disease in pregnant women or newborns. Invasive GBS disease is defined as a positive GBS culture from a normally sterile site, accompanied by related clinical manifestations.

[0050] GBS prevalence rates vary among different countries and regions. Meta-analysis showed that the GBS prevalence rate among 44,716 pregnant women reported in my country from 2000 to 2018 was 11.3%.

[0051] Without intervention, GBS colonization in pregnant women will result in a 50% chance of vertical transmission to the fetus or newborn, a significant cause of early-onset GBS disease (GBS-EOD) in newborns, which can lead to neonatal sepsis and neonatal meningitis. Therefore, establishing an effective and rapid GBS screening method for pregnant women is crucial for reducing neonatal GBS infection.

[0052] The specific RNA detection methods are as follows:

[0053] 1. Extraction of RNA from Group B Streptococcus

[0054] RNA was extracted from collected swabs or urine using our company's RNA nucleic acid extraction kit.

[0055] 2. Design of Group B Streptococcus primers and probes

[0056] This invention involves downloading the GBS 16S sequence from the NCBI database and performing homology analysis. By comparing a large number of target genes with similar sequences, regions with high conservation were selected for primer and probe design. An artificial sequence was introduced at the 5' end of the upstream primer (the bolded portion of the primer represents the introduced artificial sequence, which is not complementary to the bolded sequence in the template). The template sequence is as follows:

[0057] SEQ ID NO: 1:

[0058] CGGCAATGGACGGAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGAT

[0059] CGTAAAGCTCTGTTGTTAGAGAAGAACGTTGGTAGGAGTGGAAAATCTACCAAGTGAC

[0060] GGTAACTAACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCCCGAGCGTTTGTCCGGATTTATTG.

[0061] The primer and probe sequences are shown in Table 1.

[0062] Table 1. Primer and probe sequences

[0063]

[0064] The above primers were used to test RNA-positive samples, DNA-positive samples, and negative controls, respectively, and qRT-PCR enzyme and DNA amplification enzyme were used for testing. RNA-positive samples were diluted solutions of GBS 16s RNA transcribed using the T7 promoter; DNA-positive samples were diluted solutions of synthesized GBS 16s plasmid; and negative controls were physiological saline.

[0065] Experimental results show

[0066] DNA amplification results from the control group:

[0067] 1) The amplification efficiency of positive samples was significantly reduced after the downstream primers introduced artificial sequences, while ordinary primers could amplify positive samples normally (comparison between R1 and Ra11); 2) With the reduction of the number of complementary bases, the downstream primers with artificial sequences showed almost no amplification of positive samples (comparison between Ra11 and Ra9); 3) Using deoxyurea pyrimidine instead of deoxythymidine on the basis of Ra9 also resulted in no amplification of positive samples (comparison between Ra9 and Ra9u).

[0068] Results of qRT-PCR amplification in the experimental group (see Table 2).

[0069] 1) After introducing artificial sequences into the downstream primers, the amplification efficiency of DNA positive samples decreased significantly, while that of RNA positive samples remained unchanged. Ordinary primers could amplify both RNA and DNA positive samples normally (comparison between R1 and Ra11); 2) With the reduction of complementary bases, the amplification efficiency of DNA positive samples further decreased with the introduction of artificial sequences into the downstream primers, and that of RNA positive samples also decreased slightly (comparison between Ra11 and Ra9); 3) Using deoxyurea pyrimidine instead of deoxythymidine in Ra9 resulted in no amplification of DNA positive samples, while the efficiency of RNA positive samples increased slightly (comparison between Ra9 and Ra9u).

[0070] Table 2. Amplification Results

[0071]

[0072] 3. Preparation of PCR reaction system for Group B Streptococcus RNA detection kit

[0073] (1) Remove the kit from the refrigerator, allow it to equilibrate to room temperature, allow all components to fully dissolve, and centrifuge quickly for 5 seconds.

[0074] (2) Calculate the number of reactions required for this experiment (n = number of samples + 2T (reference standard) and prepare the reaction system according to the following table: Prepare the reaction solution according to the system in Table 3.

[0075] Table 3. GBS RNA detection reaction system configuration

[0076]

[0077] 3. Procedure and interpretation of results for the Group B Streptococcus RNA detection kit

[0078] Computer program

[0079] Follow the steps in Table 4 to set up the temperature cycling and signal acquisition program.

[0080] Table 4. Temperature Cycling and Signal Acquisition Program

[0081]

[0082] Fluorescence channel selection during detection: Select FAM or ROX fluorescence detection channels.

[0083] Interpretation of test results

[0084] Instrument settings

[0085] 1. After the reaction is complete, based on the amplification curve, determine a suitable baseline (generally set to 3 for the start and 15 for the end) and fluorescence threshold (generally set at the middle of the exponential growth phase in the logarithmic form of the amplification curve) to obtain the CT values ​​of different channels.

[0086] 2. When analyzing the results, add background signal correction and set Passive reference to None.

[0087] Quality control

[0088] A successful PCR reaction detection requires the following conditions to be met simultaneously:

[0089] Negative control: No amplification in the FAM detection channel or Ct(FAM)>38; Ct(ROX)≤35.

[0090] Positive control: The FAM detection channel has an amplification curve and Ct(FAM)≤38; the ROX detection channel may or may not have an amplification curve.

[0091] If any of the above conditions are not met, the PCR reaction is deemed to have failed, the sample test result is invalid, and the reaction must be repeated.

[0092] Result Interpretation

[0093] If the FAM detection channel has an amplification curve and Ct(FAM)≤38, and the ROX detection channel has or does not have an amplification curve, the sample can be judged as GBS positive.

[0094] If there is no amplification in the FAM detection channel or Ct(FAM) > 38; and Ct(ROX) ≤ 35 in the ROX detection channel, the sample can be judged as GBS negative.

[0095] If there is no amplification in the FAM detection channel or Ct(FAM) > 38, or no amplification in the ROX detection channel or Ct(ROX) > 35, the PCR reaction has failed. This may be due to inhibitors present during RNA sample preparation. It is recommended to dilute the RNA 10-fold or 100-fold and repeat the measurement. If there is a fluorescence signal, the result should be determined as described above. Otherwise, it is recommended to repeat the test.

[0096] 5. Experimental Results

[0097] Group B Streptococcus RNA Detection Kit Test Results for Low-Concentration Samples

[0098] The low-copy samples were tested using the method described above, such as... Figure 3 and Figure 4 As shown, this kit can accurately detect 10 3 Samples per copies / ml (Note: HBB is an internal control).

[0099] Group B Streptococcus RNA Detection Kit Specific Detection

[0100] RNA detection of other pathogens

[0101] The above methods were used to test samples of bacteria closely related to Group B Streptococcus, microorganisms that easily cause the same or similar clinical symptoms, and colonizing bacteria at the site of infection, including: Streptococcus pyogenes, Streptococcus pneumoniae, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, Herpes simplex virus, Candida albicans, and Mycoplasma hominis.

[0102] The tests showed that all strains except Group B Streptococcus were negative.

[0103] Group B Streptococcus RNA Detection Kit for GBS DNA Detection

[0104] The above methods were used to analyze 10 respectively. 6 copies / ml GBS bacterial culture and 10 copies / ml inactivated by high concentration of antibiotics 6 The GBS bacterial culture was tested at a ratio of copies / ml. Simultaneously, a comparative test was performed using a competing DNA kit (see Table 5). The results showed that the GBS bacterial culture of this invention could amplify normally, while the GBS bacterial culture inactivated by antibiotics showed no amplification signal. The competing DNA kit showed signals both before and after GBS inactivation.

[0105] Table 5. Comparison Test Results

[0106]

[0107] Group B Streptococcus RNA Detection Kit for Clinical Sample Testing

[0108] The kit was used to examine 150 clinical samples using the culture method and DNA detection method, and the results are shown in Tables 6 and 7.

[0109] Table 6. Comparison of DNA amplification method and culture method

[0110] culture method positive Culture method negative total Positive DNA amplification test 30 6 36 Negative DNA amplification test 3 111 114 total 33 117 150

[0111] Table 7. Comparison of Group B Streptococcus RNA Detection Kits and Culture Methods

[0112] culture method positive Culture method negative total Positive RNA amplification test 31 3 34 RNA amplification test negative 2 114 116 total 33 117 150

[0113] Using the culture method as the gold standard, the sensitivity of the DNA detection method was 90.9% (30 / 33), and the specificity was 94.9% (111 / 117). Using the culture method as the gold standard, the sensitivity of this Group B Streptococcus RNA detection kit was 93.9% (31 / 33), and the specificity was 97.4% (114 / 117). The results show that the RNA detection method has higher sensitivity and specificity than the DNA detection method.

[0114] Example 2:

[0115] RNA detection of Gram-negative pathogens

[0116] This example uses the detection of Neisseria gonorrhoeae RNA:

[0117] Gonorrhea is one of the major sexually transmitted diseases in my country, caused by infection with Neisseria gonorrhoeae (NG). It is characterized by a short incubation period and high infectivity; if not treated promptly, it can lead to serious complications and sequelae. Based on clinical manifestations, it is classified into symptomatic genitourinary infections (commonly manifesting as purulent inflammation), asymptomatic genitourinary infections, infections of the eyes, pharynx, skin, rectum, pelvis, etc., and hematogenous disseminated infections. Common manifestations in men include urethritis, with complications such as epididymitis, prostatitis, and seminal vesiculitis; common manifestations in women include cervicitis, urethritis, Bartholin's gland inflammation, and perianal inflammation, with complications such as pelvic inflammatory disease; it can also induce decreased fertility and neonatal conjunctivitis.

[0118] Establishing an effective and rapid method for detecting gonococcal RNA is of great significance for the treatment of gonorrhea. The specific RNA detection method is as follows:

[0119] 1. Extraction of Neisseria gonorrhoeae RNA

[0120] RNA was extracted from collected swabs or urine using our company's RNA nucleic acid extraction kit.

[0121] 2. Design of Neisseria gonorrhoeae primers and probes

[0122] This invention involves downloading the 16S sequence of Neisseria gonorrhoeae from the NCBI database and performing homology analysis. By comparing a large number of target genes with similar sequences, regions with high conservation were selected for primer and probe design. An artificial sequence was introduced at the 5' end of the upstream primer (the bolded portion of the primer represents the introduced artificial sequence, which is not complementary to the bolded sequence in the template). The template sequence is as follows:

[0123] SEQ ID NO: 8:

[0124] CCTGATCCAGCCATGCCGCGTGTCTGAAGAAGGCCTTCGGGTGTAAAGGACTTTTGTCAGGGAAGAAAAGGCTGTTGCCAATATCGGCGGCCGATGACGGT.

[0125] The primer and probe sequences are shown in Table 8.

[0126] Table 8. Primer and probe sequences

[0127] name Sequence (5'-3') NG-F (SEQ ID NO: 9) TGATCCAGCCATGCCGCGTGTC NG-Ra (SEQ ID NO: 10) ccaaCGTGATTGGTAACA / ideoxyU / AT / ideoxyU / GG NG-P (SEQ ID NO: 11) CGGGTTGTAAAGGACTTTT

[0128] 3. Preparation of PCR reaction system for gonococcal RNA detection kit

[0129] (1) Remove the kit from the refrigerator, allow it to equilibrate to room temperature, allow all components to fully dissolve, and centrifuge quickly for 5 seconds.

[0130] (2) Calculate the number of reactions required for the current experiment (n = number of samples + 2T (reference standard) and prepare the reaction system according to the following table: Prepare the reaction solution according to the system in Table 9.

[0131] Table 9. Reaction system for gonococcal RNA detection

[0132]

[0133] 4. Procedure for using the gonococcal RNA detection kit and interpretation of results

[0134] Computer program

[0135] Set up the temperature cycling and signal acquisition program according to the steps in Table 10.

[0136] Table 10. Temperature Cycling and Signal Acquisition Procedure

[0137]

[0138] Fluorescence channel selection during detection: Select FAM or ROX fluorescence detection channels.

[0139] Interpretation of test results

[0140] Instrument settings

[0141] 1. After the reaction is complete, based on the amplification curve, determine a suitable baseline (generally set to 3 for the start and 15 for the end) and fluorescence threshold (generally set at the middle of the exponential growth phase in the logarithmic form of the amplification curve) to obtain the CT values ​​of different channels.

[0142] 2. When analyzing the results, add background signal correction and set Passive reference to None.

[0143] Quality control

[0144] A successful PCR reaction detection requires the following conditions to be met simultaneously:

[0145] Negative controls: No amplification in the FAM detection channel or Ct(FAM) > 38; Ct(ROX) ≤ 35

[0146] Positive control: The FAM detection channel has an amplification curve and Ct(FAM)≤38; the ROX detection channel may or may not have an amplification curve.

[0147] If any of the above conditions are not met, the PCR reaction is deemed to have failed, the sample test result is invalid, and the reaction must be repeated.

[0148] Result Interpretation

[0149] If the FAM detection channel shows an amplification curve and Ct(FAM)≤38, and the ROX detection channel shows or does not show an amplification curve, the sample can be judged as positive for Neisseria gonorrhoeae.

[0150] If there is no amplification in the FAM detection channel or Ct(FAM) > 38; and Ct(ROX) ≤ 35 in the ROX detection channel, the sample can be judged as negative for gonococci.

[0151] If there is no amplification in the FAM detection channel or Ct(FAM) > 38, or no amplification in the ROX detection channel or Ct(ROX) > 35, the PCR reaction has failed. This may be due to inhibitors present during RNA sample preparation. It is recommended to dilute the RNA 10-fold or 100-fold and repeat the measurement. If there is a fluorescence signal, the result should be determined as described above. Otherwise, it is recommended to repeat the test.

[0152] Experimental results

[0153] Gonococcal RNA detection kit test results for low concentration samples

[0154] The low-copy samples were tested using the method described above, such as... Figure 5 and Figure 6 As shown, this kit can accurately detect 10 2 Samples per 100 copies / ml (Note: HBB is an internal control).

[0155] Specific detection of Neisseria gonorrhoeae RNA detection kit

[0156] RNA detection of other pathogens

[0157] The following samples were tested using the above method: closely related bacteria to Neisseria gonorrhoeae, microorganisms that easily cause the same or similar clinical symptoms, and colonizing bacteria at the site of infection: Streptococcus, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Chlamydia trachomatis, Ureaplasma urealyticum, Herpes simplex virus, Candida albicans, and Mycoplasma hominis.

[0158] The tests showed that all strains except Neisseria gonorrhoeae were negative.

[0159] Gonococcal RNA detection kit for detecting gonococcal DNA

[0160] The above methods were used to analyze 10 respectively. 6 Copies / ml of gonococci and 10 copies / ml of gonococci inactivated by high-concentration antibiotics 6 The test was performed using copies / ml of Neisseria gonorrhoeae. A comparative test was also conducted using a competing DNA kit (see Table 11). The results showed that the present invention could detect and amplify Neisseria gonorrhoeae normally, while Neisseria gonorrhoeae inactivated by antibiotics showed no amplification signal. The competing DNA kit showed signals for Neisseria gonorrhoeae both before and after inactivation.

[0161] Table 11. Comparison Test Results

[0162]

[0163] Example 3

[0164] Fungal pathogen RNA detection

[0165] This example uses the detection of Candida albicans RNA:

[0166] Candidiasis is an acute, subacute, or chronic infection primarily caused by Candida albicans, and is the most common fungal infection. It commonly affects the skin and mucous membranes, but can also cause visceral or systemic infections. Clinical symptoms are complex and vary in severity. In children, it is often an acute secondary infection. In recent years, with the use of high-dose antibiotics, hormones, and immunosuppressants, as well as the development of organ transplantation, its incidence has been gradually increasing, and it can be life-threatening with serious consequences.

[0167] Current methods for detecting fungal antigens and antibodies or metabolites using microscopy, culture, and serological methods lack sufficient sensitivity and specificity. Early detection and treatment can reduce mortality; therefore, establishing a rapid and accurate method for early detection of fungal infections is crucial. Specific RNA detection methods are as follows:

[0168] 1. Extraction of RNA from Candida albicans

[0169] RNA was extracted from collected swabs or urine using our company's RNA nucleic acid extraction kit.

[0170] 2. Design of primers and probes for Candida albicans

[0171] This invention involves downloading the 18S sequence of *Candida albicans* from the NCBI database and performing homology analysis. By comparing a large number of target genes with similar sequences, regions with high conservation were selected for primer and probe design. An artificial sequence was introduced at the 5' end of the upstream primer (the bolded portion of the primer represents the introduced artificial sequence, which is not complementary to the bolded sequence in the template). The template sequence is as follows:

[0172] SEQ ID NO: 12:

[0173] CAAGAACGAAAGTTAGGGGATCGAAGATGATCAGATACCGTCGTAGTCTTAACCAT AAACTATGCCGACTAGGGATCGGTTGTTGTTCTTTTATTGACGCAATCGGCACCTTACGA GAAATCAAAGTCTTTGGGTTCTGGGGGGAGTATGGTCGCAAGGCTGAAACTT.

[0174] The primer and probe sequences are shown in Table 12.

[0175] Table 12. Primer and probe sequences

[0176]

[0177] 3. Preparation of PCR reaction system for Candida albicans RNA detection kit

[0178] (1) Remove the kit from the refrigerator, allow it to equilibrate to room temperature, allow all components to fully dissolve, and centrifuge quickly for 5 seconds.

[0179] (2) Calculate the number of reactions required for the current experiment (n = number of samples + 2T (reference standard) and prepare the reaction system according to the following table: Prepare the reaction solution according to the system in Table 13.

[0180] Table 13. Reaction system for Candida albicans RNA detection

[0181]

[0182]

[0183] Procedures and interpretation of results for the Candida albicans RNA detection kit

[0184] Computer program

[0185] Follow the steps in Table 14 to set up the temperature cycling and signal acquisition program.

[0186] Table 14. Temperature Cycling and Signal Acquisition Program

[0187]

[0188] Fluorescence channel selection during detection: Select FAM or ROX fluorescence detection channels.

[0189] Interpretation of test results

[0190] Instrument settings

[0191] 1. After the reaction is complete, based on the amplification curve, determine a suitable baseline (generally set to 3 for the start and 15 for the end) and fluorescence threshold (generally set at the middle of the exponential growth phase in the logarithmic form of the amplification curve) to obtain the CT values ​​of different channels.

[0192] 2. When analyzing the results, add background signal correction and set Passive reference to None.

[0193] Quality control

[0194] A successful PCR reaction detection requires the following conditions to be met simultaneously:

[0195] Negative control: No amplification in the FAM detection channel or Ct(FAM)>38; Ct(ROX)≤35.

[0196] Positive control: The FAM detection channel has an amplification curve and Ct(FAM)≤38; the ROX detection channel may or may not have an amplification curve.

[0197] If any of the above conditions are not met, the PCR reaction is deemed to have failed, the sample test result is invalid, and the reaction must be repeated.

[0198] Result Interpretation

[0199] If the FAM detection channel shows an amplification curve and Ct(FAM)≤38, and the ROX detection channel shows or does not show an amplification curve, the sample can be judged to be positive for Candida albicans.

[0200] If there is no amplification in the FAM detection channel or Ct(FAM)>38; and Ct(ROX)≤35 in the ROX detection channel, the sample can be judged as negative for Candida albicans.

[0201] If the FAM detection channel shows no amplification or Ct(FAM) > 38, or the ROX detection channel shows no amplification or Ct(ROX) > 35, the PCR reaction has failed. This may be due to inhibitors present during RNA sample preparation. It is recommended to dilute the RNA 10-fold or 100-fold and repeat the measurement. If a fluorescence signal is detected, proceed as described above; otherwise, it is recommended to repeat the reaction.

[0202] Experimental results

[0203] Candida albicans RNA detection kit detects low concentration samples.

[0204] The low-copy samples were tested using the method described above, such as... Figure 7 and Figure 8 As shown, this kit can accurately detect 10 3 Samples per 100 copies / ml (Note: HBB is an internal control).

[0205] Candida albicans RNA detection kit for specific detection

[0206] RNA detection of other pathogens

[0207] The following samples were tested using the above method: closely related bacteria to Candida albicans, microorganisms that easily cause the same or similar clinical symptoms, and colonizing bacteria at the site of infection: Trichomonas vaginalis, Streptococcus, Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Neisseria gonorrhoeae, Chlamydia trachomatis, Ureaplasma urealyticum, Neisseria gonorrhoeae, Herpes simplex virus, and Mycoplasma hominis.

[0208] The tests showed that all strains except for Candida albicans were negative.

[0209] Candida albicans RNA detection kit for DNA detection

[0210] The above methods were used to analyze 10 respectively. 6 Copies / ml of Candida albicans culture and 10 copies / ml of Candida albicans inactivated by high concentration of antibiotics 6 The samples were tested at 1 / ml copies / ml of Candida albicans bacterial suspension. Simultaneously, a comparative test was performed using a competing DNA kit (see Table 15). The results showed that the Candida albicans bacterial suspension of this invention could amplify normally, while the Candida albicans bacterial suspension inactivated by antibiotics showed no amplification signal. The competing DNA kit showed signals before and after Candida albicans inactivation.

[0211] Table 15. Comparison Test Results

[0212]

[0213] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A nucleic acid detection kit for distinguishing Group B Streptococcus DNA / RNA, characterized in that, The kit includes primers and probes. The sequences of the downstream primers are shown in SEQ ID NO: 4, SEQ ID NO: 5 or cgagcATCATTACCAACGC / ideoxyU / CG, the sequences of the upstream primers are shown in SEQ ID NO: 2, and the sequences of the probes are shown in SEQ ID NO:

7.

2. A nucleic acid detection kit for distinguishing Neisseria gonorrhoeae DNA / RNA, characterized in that, The kit includes primers and probes. The sequence of the downstream primer is shown as ccaaCGTGATTGGTAACA / ideoxyU / AT / ideoxyU / GG, the sequence of the upstream primer is shown as SEQ ID NO: 9, and the sequence of the probe is shown as SEQ ID NO:

11.

3. A nucleic acid detection kit for distinguishing between Candida albicans DNA / RNA, characterized in that, The kit includes primers and probes. The sequence of the downstream primer is shown as tatgCAGAGTTGAGATCGACCA / ideoxyU / A, the sequence of the upstream primer is shown as SEQ ID NO: 13, and the sequence of the probe is shown as SEQ ID NO: 15.

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